Rap1-mediated nuclear factor-kappaB (NF-kappa B) activity regulates the paracrine capacity of mesenchymal stem cells in heart repair following infarction

Paracrine effect is the major mechanism that underlies mesenchymal stem cells (MSC)-based therapy. This study aimed to examine how Rap1, telomeric repeat-binding factor 2-interacting protein 1 (Terf2IP), which is a novel modulator involved in the nuclear factor-kappaB (NF-kappa B) pathway, regulates the paracrine effects of MSC-mediated heart repair following infarction. NF-kappa B activity of stromal cells was increased by Rap1 as measured by pNF-kappa B-luciferase reporter activity, and this was abolished by IkB-dominantnegative protein. Knockdown of Rapt with shRap1 resulted in diminished translocation of p65-NF-kappa B from the cytoplasm to nuclei in response to tumor necrosis factor-a (TNF-alpha) stimulation. Compared with BM-MSCs, Rap1(-/-) -BM-MSCs displayed a significantly reduced ratio of phosphorylated NF-kappa B to NF-kappa B-p65 and of Bax to Bcl-2, and increased resistance to hypoxia-induced apoptosis by the terminal deoxynucleotidal transferase-mediated dUTP nick end labeling (TUNEL) assay. In contrast, re-expression of Rap1 in Rap1(-/-) -BM-MSCs resulted in loss of resistance to apoptosis in the presence of hypoxia. Moreover, absence of Rap1(-/-) in BM-MSCs led to downregulation of NF-kappa B activity accompanied by reduced pro-inflammatory paracrine cytokines TNF-alpha, IL (interleukin)-6 and monocyte chemotactic protein-1 in Rap1(-/-) -BM-MSCs compared with BM-MSCs. The apoptosis of neonatal cardiomyocytes (NCMCs) induced by hypoxia was significantly reduced when cocultured with Rapt1(-/-) -BM-MSC hypoxic-conditioned medium (CdM). The increased cardioprotective effects of Rapt -BM-MSCs were reduced when Rap1(-/-) -BM-MSCs were reconstituted with Rap1(-/-) re-expression. Furthermore, in vivo study showed that transplantation of Rap1(-/-) -BM-MSCs significantly improved heart function, decreased infarct size, prevented cardiomyocyte apoptosis and inhibited inflammation compared with controls and BM-MSCs (P < 0.01). This study reveals that Rap1(-/-) has a critical role in the regulation of MSC paracrine actions. Compared with BM-MSCs, Rap1(-/-) -BM-MSCs decreased NF-kappa B sensitivity to stress-induced pro-inflammatory cytokine production and reduced apoptosis. Selective inhibition of Rap1 in BM-MSCs may be a novel strategy to enhance MSC-based therapeutic efficacy in myocardial infarction.